T cell receptor targeting AFP peptide, its preparation method and companion diagnostic kit

Abstract

The present invention relates to the field of biomedical technologies, and particularly to a T cell receptor targeting an AFP peptide, a preparation method thereof, and an accompanying diagnostic kit. A T cell receptor targeting an AFP peptide is provided, the sequence of the AFP peptide is FMNKFIYEI, and the T cell receptor comprises a variable domain of the TCRα chain and a variable domain of the TCRβ chain. Corresponding nucleic acid molecules, vectors, and host cells are provided, as well as a method for preparing the T cell receptor. An accompanying diagnostic kit containing the cell receptor is further provided. The present invention provides a better choice for products for the accompanying diagnosis and treatment of cells of alpha-fetoprotein (AFP).

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a T cell receptor targeting AFP peptide, a preparation method thereof, and an accompanying diagnostic kit. Background Art

[0002] The adoptive T cell therapy (ACT) strategy has become a very important treatment strategy in cancer immunotherapy. Engineered T cell receptor-T cell (TCR-T) therapy and chimeric antigens receptor-T cell (CAR-T) therapy are two main methods for specifically redirecting T cells to tumor antigens. Novartis' Kymriah is a CD19-directed CAR-T cell immunotherapy and is also the world's first CAR-T therapy approved by the FDA for marketing. Currently, more than a dozen CAR-T products have been approved for marketing globally. From the perspective of the current research situation, although CAR-T therapy has significant efficacy in the treatment of B cell malignancies, its treatment of solid tumors has not achieved satisfactory results.

[0003] TCR-T is a T cell expressing a tumor antigen-specific receptor, and its α-chain and β-chain are generated from high-quality and high-affinity antigen-specific T cell clones. In recent years, TCR-T cell therapy has made a series of progress, mainly used for the treatment of solid tumors. The types of tumor antigens covered include tumor mutant neoantigens (e.g., KRAS mutant antigen), tissue differentiation antigens (e.g., gp100), cancer-testis antigens (e.g., MAGE-A4), viral antigens (e.g., HPV-E6), overexpressed antigens (e.g., HER2), etc.; currently, more than 150 clinical trials are underway; on January 31, 2024, Adaptimmune announced that the biologics license application (BLA) of its TCR-T therapy Afami-cel was accepted by the FDA and obtained priority review status, and it was approved for marketing on August 1, 2024.

[0004] Compared with chimeric antigen receptor (CAR), T cell receptor (TCR) has some obvious advantages in T cell-based therapies, mainly including: 1. There are more subunits in its receptor structure, and there are more immunoreceptor tyrosine-based activation motifs (ITAMs), with less dependence on antigens; 2. There are more co-stimulatory receptors (CD3, CD4, CD28, etc.), which can enable TCRs with a low MHC affinity range to effectively activate T cells; 3. Since it can target intracellular tumor-specific antigens, TCR has more potential in the treatment of solid tumors.

[0005] Alpha-fetoprotein (AFP) is a glycoprotein that belongs to the albumin family. It is mainly synthesized by fetal hepatocytes and yolk sacs. During fetal development, AFP mainly plays a transport role, carrying substances such as fatty acids and bilirubin. In addition, it also has an immunomodulatory function, protecting the fetus from the attack of the maternal immune system. The concentration of AFP in the blood of healthy adults is extremely low, and it is mainly produced in small amounts in the liver. Research shows that in about 50% to 80% of patients with hepatocellular carcinoma (HCC), the transcription of AFP is reactivated and highly expressed, becoming a protein that is specifically highly expressed in liver cancer cells. HCC patients infected with HBV, which is the most common in the Chinese population, often have a high degree of malignancy and a high level of AFP expression. Comprehensive current research shows that AFP is an attractive target for adoptive T cell therapy for HCC, especially for patients with a high degree of malignancy and limited treatment options.

[0006] In addition to the treatment field, TCR technology can also be used for the companion diagnosis of cancer immunotherapy. For example, after screening and obtaining TCR molecules against specific mutant peptides, they can be used to detect the cloning and identification of T cell receptor genes in the tissue samples of subjects. By detecting the cloning and identification of T cell receptor genes in the samples of the subjects at different time points, comprehensively analyzing relevant TCR cloning ratios, cloning diversities and other indicators, and real-time monitoring of the dynamic changes of sample TCR indicators, such as the total number of clones, clone types, diversity indices, clone abundances, etc., to assist in evaluating the clinical efficacy of immunotherapy and screening patient populations with better responsiveness and higher clinical benefits.

[0007] Therefore, in order to conduct relevant companion diagnoses more conveniently, TCR receptor proteins can be prepared into kits for the companion diagnosis of subjects.

[0008] Currently, there are not many companion diagnostic and therapeutic products for cells targeting alpha-fetoprotein (AFP). Currently, there are only more than a dozen TCR-T products under research, all in the clinical phase I. Therefore, for products targeting this target, patients need more diverse options that can achieve greater clinical benefits. Summary of the Invention

[0009] The technical problem to be solved by the present invention is that there are not many companion diagnostic and therapeutic products for cells targeting alpha-fetoprotein (AFP) currently.

[0010] Accordingly, the technical solution adopted by the present invention to solve its technical problems is to screen and obtain a T cell receptor specifically targeting AFP peptide and use it in an adjunct diagnostic kit. Specifically, some AFP peptide segments (such as AFP158-166) can bind to specific HLA molecules such as HLA-A*02:01, form an antigen peptide-MHC complex and be recognized by TCR, inducing an anti-tumor immune response. By using the engineered AFP-specific TCR protein to detect the presence of AFP / HLA complex on the surface of tumor tissues or cells, it is expected to be used to screen the suitable population for AFP-targeted TCR-T cell therapy or AFP vaccine therapy, as an adjunct diagnostic tool for related immunotherapy products.

[0011] In a first aspect, there is provided a T cell receptor targeting an AFP peptide, the sequence of the AFP peptide being FMNKFIYEI (SEQ ID NO:25), and the T cell receptor comprising a variable domain of the TCRα chain and a variable domain of the TCRβ chain.

[0012] Wherein, the variable domain of the TCRα chain comprises:

[0013] CDR1α with the sequence DSASNY (SEQ ID NO:1);

[0014] CDR2α with the sequence IRSNVGE (SEQ ID NO:2); and

[0015] CDR3α with the sequence AAPFGGEAGTALI (SEQ ID NO:3).

[0016] Wherein, the variable domain of the TCRβ chain comprises:

[0017] CDR1β with the sequence DFQATT (SEQ ID NO:4);

[0018] CDR2β with the sequence SNEGSKA (SEQ ID NO:5); and

[0019] CDR3β with the sequence SARGPLIGPTDTQY (SEQ ID NO:6).

[0020] In some embodiments, the T cell receptor is soluble.

[0021] In some embodiments, an artificial disulfide bond is included between the constant region of the α chain and the constant region of the β chain of the T cell receptor.

[0022] In some embodiments, the sequence of the variable domain of the TCRα chain is:

[0023] MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAAPFGGEAGTALIFGKGTTLSVSSN (SEQ ID NO:19),

[0024] The sequence of the variable region of the TCR β chain is:

[0025] MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARGPLIGPTDTQYFGPGTRLTVLE (SEQ ID NO:20).

[0026] In some embodiments, the T cell receptor is a fusion protein.

[0027] In a second aspect, there is provided a synthetic nucleic acid molecule encoding the T cell receptor of the first aspect of the present invention.

[0028] In a third aspect, there is provided a vector containing the synthetic nucleic acid molecule of the present invention.

[0029] In a fourth aspect, there is provided a host cell containing the vector of the present invention.

[0030] Alternatively, there is provided a host cell in which the synthetic nucleic acid molecule of the present invention is integrated into the chromosome.

[0031] In a fifth aspect, a method for preparing a T cell receptor includes:

[0032] (i) culturing the host cell of the present invention to express the T cell receptor of the first aspect of the present invention; and

[0033] (ii) isolating the T cell receptor.

[0034] In a sixth aspect, there is provided an adjuvant diagnostic kit for tumor immunotherapy, including:

[0035] A T cell receptor protein comprising a variable region of the TCR α chain and a variable region of the TCR β chain,

[0036] The variable region of the TCR α chain comprises:

[0037] CDR1α with the sequence DSASNY (SEQ ID NO:1);

[0038] CDR2α with the sequence IRSNVGE (SEQ ID NO:2); and

[0039] CDR3α with the sequence AAPFGGEAGTALI (SEQ ID NO:3),

[0040] wherein the variable domain of the TCRβ chain comprises:

[0041] CDR1β with the sequence DFQATT (SEQ ID NO:4);

[0042] CDR2β with the sequence SNEGSKA (SEQ ID NO:5); and

[0043] CDR3β with the sequence SARGPLIGPTDTQY (SEQ ID NO:6), wherein the T cell immune receptor targets a tumor antigen peptide derived from the AFP gene, and the sequence of the tumor antigen peptide is: FMNKFIYEI.

[0044] In some embodiments, the kit further comprises a chromogenic system, a positive control, a negative control, a staining buffer, and a blocking solution.

[0045] In some embodiments, the samples of the companion diagnostic kit include: tissue samples, peripheral blood, and tumor-infiltrating TILs.

[0046] In some embodiments, the companion diagnostic kit includes: an immunohistofluorescence staining (IHC) kit or a flow cytometry detection kit.

[0047] In some embodiments, the method of using the companion diagnostic kit includes:

[0048] 1). Staining tumor tissue sections with the TCR-Fc protein probe;

[0049] 2). Detecting the presence of the AFP / HLA complex;

[0050] 3). Evaluating the AFP antigen presentation ability according to the detection result.

[0051] In some embodiments, the method of using the companion diagnostic kit includes:

[0052] 1). Using the TCR protein probe for flow cytometry staining;

[0053] 2). Detecting whether the AFP / HLA complex is expressed on the cell surface;

[0054] 3). Performing dynamic immune monitoring by evaluating the antigen presentation level or AFP expression.

[0055] In a seventh aspect, the present invention relates to a composition comprising a fusion polypeptide, the fusion polypeptide comprising the aforementioned TCR α-chain and / or TCR β-chain.

[0056] In some embodiments, a cell is genetically modified by introducing a isolated nucleic acid molecule encoding a polypeptide comprising at least one of the aforementioned TCR α-chain and TCR β-chain.

[0057] In some embodiments, the cell is an immune cell.

[0058] In some embodiments, the immune cells are selected from the group consisting of: antigen-presenting cells, B cells, dendritic cells, macrophages, Langerhans cells, T cells, NK cells, NK T cells.

[0059] The beneficial effect of the present invention is that by screening with AFP peptide, a T cell receptor with high affinity for AFP peptide and stable expression on the cell membrane is obtained. This T cell receptor can specifically bind to the FMNKFIYEI-HLA-A*02:01 complex, providing a basis for the companion diagnosis of tumor immunotherapy. In addition, this T cell receptor exhibits excellent specific killing activity against antigen-positive tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 A graph showing the experimental results of the binding affinity of TCR for AFP peptide.

[0061] Figure 2 A graph showing the experimental results of the stability of TCR expression on the cell membrane.

[0062] Figure 3 A graph showing the experimental results of the effect of T cells overexpressing TCR on the specific IFN-γ secretion of antigen-positive target cells.

[0063] Figure 4 A graph showing the experimental results of the effect of T cells overexpressing TCR on the specific IL-2 secretion of antigen-positive target cells.

[0064] Figure 5 A graph showing the experimental results of the effect of T cells overexpressing TCR on CD137 expression.

[0065] Figure 6 A graph showing the experimental results of the specific killing activity of T cells overexpressing TCR against antigen-positive tumor cells. DETAILED DESCRIPTION OF THE INVENTION

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0067] As used herein, "CDR" is defined as the amino acid sequence of the complementarity determining region of a TCR or TCR chain.

[0068] In the context of the present invention, the following abbreviations for common nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0069] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up the sequence of the protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and long chains (which are commonly referred to in the art as proteins, and there are many types of proteins).

[0070] As used herein, "vector" can mean a nucleic acid sequence containing an origin of replication. The vector can be a plasmid, phage, bacterial artificial chromosome, or yeast artificial chromosome. The vector can be a DNA or RNA vector. The vector can be an extrachromosomal vector that self-replicates or a vector integrated into the host genome.

[0071] In some embodiments, the TCR comprises a variable domain of the TCRα chain and a variable domain of the TCRβ chain. The 3 complementarity determining regions (CDRs) of the variable domain of the TCRα chain are:

[0072] CDR1α - DSASNY (SEQ ID NO:1);

[0073] CDR2α - IRSNVGE (SEQ ID NO:2); and

[0074] CDR3α - AAPFGGEAGTALI (SEQ ID NO:3), and

[0075] The 3 complementarity determining regions (CDRs) of the variable domain of the TCRβ chain are:

[0076] CDR1β - DFQATT (SEQ ID NO:4);

[0077] CDR2β - SNEGSKA (SEQ ID NO:5); and

[0078] CDR3β - SARGPLIGPTDTQY (SEQ ID NO:6).

[0079] In some embodiments, the TCR comprises a variable domain of the TCR α-chain and a variable domain of the TCR β-chain. The three complementarity-determining regions (CDRs) of the variable domain of the TCR α-chain are:

[0080] CDR1α - DRVSQS (SEQ ID NO:7);

[0081] CDR2α - IYSNGD (SEQ ID NO:8); and

[0082] CDR3α - AVTPGSNYKLT (SEQ ID NO:9), and

[0083] The three complementarity-determining regions (CDRs) of the variable domain of the TCR β-chain are:

[0084] CDR1β - SGHNT (SEQ ID NO:10);

[0085] CDR2β - YYREEE (SEQ ID NO:11); and

[0086] CDR3β - ASSPGLAGEQY (SEQ ID NO:12).

[0087] In some embodiments, the TCR comprises a variable domain of the TCR α-chain and a variable domain of the TCR β-chain. The three complementarity-determining regions (CDRs) of the variable domain of the TCR α-chain are:

[0088] CDR1α - DPNSYY (SEQ ID NO:13);

[0089] CDR2α - VFSSTEI (SEQ ID NO:14); and

[0090] CDR3α - AVNSNYAQGLT (SEQ ID NO:15), and

[0091] The three complementarity-determining regions (CDRs) of the variable domain of the TCR β-chain are:

[0092] CDR1β - NSQYPW (SEQ ID NO:16);

[0093] CDR2β - LRSPGD (SEQ ID NO:17); and

[0094] CDR3β - TCSGSGGAETLY (SEQ ID NO:18).

[0095] In some embodiments, the TCR comprises an α - chain variable domain amino acid sequence as shown in SEQ ID NO:19, and the TCR comprises a β - chain variable domain amino acid sequence as shown in SEQ ID NO:20.

[0096] In some embodiments, the TCR comprises an α - chain variable domain amino acid sequence as shown in SEQ ID NO:21, and the TCR comprises a β - chain variable domain amino acid sequence as shown in SEQ ID NO:22.

[0097] In some embodiments, the TCR comprises an α - chain variable domain amino acid sequence as shown in SEQ ID NO:23, and the TCR comprises a β - chain variable domain amino acid sequence as shown in SEQ ID NO:24.

[0098] In some embodiments, the TCR is single - chain.

[0099] In some embodiments, the TCR is formed by connecting the α - chain variable region and the β - chain variable region through a peptide - linking sequence.

[0100] In some embodiments, cysteine residues form an artificial disulfide bond between the constant domains of the α and β chains of the TCR.

[0101] In some embodiments, a conjugate is bound to the C - or N - terminus of the α - chain and / or β - chain of the TCR. Preferably, the conjugate is a detectable label, a therapeutic agent, a PK - modifying moiety, or a combination of any of these substances.

[0102] In some embodiments, the TCR is a murine TCR, a human - murine chimeric TCR, or a humanized TCR.

[0103] In some embodiments, the vector includes an expression vector, i.e., a construct capable of expressing in vivo or in vitro. Commonly used vectors include bacterial plasmids, phages, and viral vectors.

[0104] In some embodiments, viral vectors include, but are not limited to: adenoviral vectors, adeno - associated virus (AAV) vectors, herpesvirus vectors, retroviral vectors, lentiviral vectors, baculoviral vectors. Preferably, the vector can transfer the nucleotide of the present invention into cells, such as T cells, so that the cells express the AFP - antigen - specific TCR. The vector should be able to express at a continuously high level in T cells.

[0105] In some embodiments, the lentiviral vector may include: the lentiviral expression vector pLenti (addgene).

[0106] In some embodiments, the host cell is a mammalian cell. For example, the host cell is a human cell. Although the host cell can be a cell of any cell type, can be derived from any type of tissue, and can be a cell at any developmental stage, the host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell.

[0107] In some embodiments, when the host cell or related cell population is administered, the host cell can be a cell that is allogeneic or autologous to the mammal. Preferably, the cell is autologous to the mammal.

[0108] In some embodiments, "mammal" refers to any mammal, including but not limited to: mammals of the order Rodentia such as mice and hamsters, and mammals of the order Lagomorpha such as rabbits; preferably the mammal is from the order Carnivora, including felines (cats) and canines (dogs). More preferably the mammal is from the order Artiodactyla including bovines (cows) and suids (pigs), or from the order Perissodactyla including equines (horses); most preferably the mammal is from the order Primates, the suborder Haplorhini or the suborder Simiiformes, or from the suborder Anthropoidea (humans and anthropoid apes). Particularly preferably the mammal is a human.

[0109] Specific embodiments are used herein to illustrate the principles and implementation manners of the present invention. The description of the embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0110] Example 1: Cloning of antigenic short peptide-specific T cells

[0111] Peripheral blood lymphocytes (PBL) from healthy volunteers with the genotype HLA-A*02:01 were stimulated separately or in combination with synthetic short peptides (Genescript) AFP (SEQ ID NO:25). The above short peptides were renatured with biotin-labeled HLA-A*02:01 respectively to prepare pHLA haploids. These haploids were combined with streptavidin labeled with PE (BD) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double-positive cells were sorted using a BD Melody flow sorting instrument, and the positive cells were sorted into a 96-well plate, with one cell in each well.

[0112] Example 2: Acquisition of the TCR gene of AFP antigenic short peptide-specific T cell clones and construction of vectors

[0113] The cells obtained in Example 1 were lysed with 0.1% Triton-X (Sangon Biotech), and then the SMARTRACE cDNA amplification kit from Clontech was used. The primers were designed in the C-terminal conserved region of the human TCR gene. Downstream primer for the conserved region of the C segment of the TCR α chain: tcagctggaccacagc (SEQ ID NO:26); Downstream primer for the conserved region of the C segment of the TCR β chain: tcagaaatcctttctcttgac (SEQ ID NO:27). The full-length genes of the TCR α chain and β chain were cloned into the lentiviral expression vector pCDH (SBI) by overlap PCR respectively. The specific steps were as follows: The full-length genes of the TCR α chain and TCR β chain were ligated by overlap PCR to obtain the TCR α-2A-TCR β fragment. The lentiviral expression vector and TCR α-2A-TCR β were digested and ligated to obtain the pCDH-TRA-2A-TRB plasmid, which was sequenced and confirmed (IMGT) to obtain plasmids of three TCR clones: Y2386-5, Y2357-7, and Y24081-2.

[0114] The sequencing results of CDR1α, CDR2α, and CDR3α in the variable domain of the TCR α chain of Y2386-5 were SEQ ID NO:1-3 in sequence; the sequencing results of CDR1β, CDR2β, and CDR3β in the variable domain of the TCR β chain were SEQ ID NO:4-6 in sequence. The sequencing result of the variable domain of the TCR α chain was SEQ ID NO:19, and the sequencing result of the variable domain of the TCR β chain was SEQ ID NO:20.

[0115] The sequencing results of CDR1α, CDR2α, and CDR3α in the variable domain of the TCR α chain of Y2357-7 were SEQ ID NO:7-9 in sequence; the sequencing results of CDR1β, CDR2β, and CDR3β in the variable domain of the TCR β chain were SEQ ID NO:10-12 in sequence. The sequencing result of the variable domain of the TCR α chain was SEQ ID NO:21, and the sequencing result of the variable domain of the TCR β chain was SEQ ID NO:22.

[0116] The sequencing results of CDR1α, CDR2α, and CDR3α in the variable domain of the TCR α chain of Y24081-2 were SEQ ID NO:13-15 in sequence; the sequencing results of CDR1β, CDR2β, and CDR3β in the variable domain of the TCR β chain were SEQ ID NO:16-18 in sequence. The sequencing result of the variable domain of the TCR α chain was SEQ ID NO:23, and the sequencing result of the variable domain of the TCR β chain was SEQ ID NO:24.

[0117] Subsequently, 293T cells were used to package pseudoviruses. Specifically, the above plasmids were mixed with VSVG plasmid, RRE plasmid, and Rev plasmid (purchased from Addgene) at a ratio of 4:5:4:10, and 20 μL was taken and diluted into DMEM medium (1.25 mL) to obtain a DNA solution. 20 μL of polyetherimide (PEI 1 μg / μL) was added to DMEM (1.25 mL), and the above PEI / DMEM mixed solution was added to the prepared DNA solution. After incubating at room temperature for 20 minutes, it was added to 293T cells cultured in a 15 cm dish and mixed evenly. After 6 hours, the fresh DMEM medium was replaced. After 72 hours, the supernatant containing lentivirus was collected, which was the lentivirus supernatant of each TCR.

[0118] Example 3: Construction of overexpression cell lines of antigenic short peptide-specific TCR

[0119] The NFAT-GFP element (Addgene) was synthesized and inserted into the expression vector pCDH(SBI) by the standard method described in "Molecular Cloning: A Laboratory Manual". After the fragment was sequenced and confirmed to be correct, 293T (Procell CL-0130) was used to package pseudoviruses. The Jurkat cell line was infected with pseudoviruses containing the NFAT-GFP element and pseudoviruses containing the TCR element. Through limited dilution and monoclonal amplification, each Jurkat-NFAT-GFP-TCR overexpression cell line was obtained, namely: Jurkat-NFAT-GFP-Y2386-5-TCR, Jurkat-NFAT-GFP-Y2357-7-TCR, and Jurkat-NFAT-GFP-Y24081-2-TCR.

[0120] Example 4: Binding affinity experiment of TCR to mutant peptide, wild-type peptide or tumor-associated antigen peptide

[0121] (1) Construction of K562CD80-HLA-A*02:01 and 293T-CD80-HLA-A*02:01

[0122] The HLA-A*02:01 element (IMGT / HLA Acc No: HLA00043) and CD80 (NP_005182.1) were synthesized and inserted into the expression vector pCDH(SBI). After the fragment was sequenced and confirmed to be correct, lentivirus was packaged using the 293T cell line. Specifically, the plasmid containing the CD80-HLA-A*02:01 element was mixed with the VSVG plasmid, RRE plasmid, and Rev plasmid (purchased from Addgene) at a ratio of 4:5:4:10, and 20 μL was diluted into DMEM medium (1.25 mL) to prepare a DNA solution. 20 μL of polyetherimide (PEI 1 μg / μL) was added to DMEM (1.25 mL), and the above PEI / DMEM mixed solution was added to the prepared DNA solution. After incubating at room temperature for 20 minutes, it was added to the 293T cells cultured in a 15 cm dish and mixed evenly. After 6 hours, the fresh DMEM medium was replaced. After 72 hours, the supernatant containing lentivirus was collected, which was the lentivirus supernatant of CD80-HLA-A*02:01. The K562 or 293T cell line was infected with the pseudovirus containing the CD80-HLA-A*02:01 element, and through limited dilution and monoclonal amplification, the K562-CD80-HLA-A*02:01 and 293T-CD80-HLA-A*02:01 overexpressing cell lines were obtained.

[0123] (2) Co-culture of Jurkat-NFAT-GFP-TCR overexpressing cell line and K562-CD80-HLA-A*02:01

[0124] The cell lines (Jurkat-NFAT-GFP-Y2386-5-TCR, Jurkat-NFAT-GFP-Y2357-7-TCR, and Jurkat-NFAT-GFP-Y24081-2-TCR) were co-incubated with K562-CD80-HLA-A*02:01 loaded with different concentrations of the antigen peptide to be tested (FMNKFIYEI (AFP)). Specifically, K562 was incubated with different concentrations of the target antigen peptide in an incubator at 37°C for 1 h. After centrifugation, the cells were resuspended with the medium. The Jurkat and K562 cells were counted separately. 2×10^4 cells of Jurkat and K562 loaded with the polypeptide were aspirated and mixed, and then co-cultured in a 96-well plate. After 24 h of co-culture, the activation level of the reporter gene of Jurkat cells and the activation level of CD69 cells were detected by flow cytometry.

[0125] The results are as Figure 1Shown: TCR-T cells expressing Y2386-5, Y2357-7, and Y24081-2 have strong reactivity and specificity to the target antigen peptide AFP, and are suitable for using the related T cell receptor proteins for diagnostic and therapeutic purposes.

[0126] Example 5: Experiment on the stability of TCR overexpression in cell membrane expression

[0127] TCR-T cells (Y23191, Y23192, and Y24318) expressing TCR were constructed using the TCR elements (TCR α-2A-TCR β fragment) of Y2386-5, Y2357-7, and Y24081-2 respectively. The specific steps are as follows:

[0128] (1) Preparation of TCR lentivirus: Each TCR element and GFP (addgene) were synthesized and inserted into the expression vector pCDH(SBI) by the standard method described in "Molecular Cloning a Laboratory Manual" (ISBN 978-1-936113-42-2; Chapter 3 Cloning and Transformation with Plasmid Vectors). After the fragments were sequenced and confirmed to be correct, 293T (Procell CL-0130) was used to package pseudovirus according to the specific operation steps of Example 2.

[0129] (2) Construction of TCR-T cells expressing TCR: After thawing PBMC, use an appropriate amount of X-VIVO 15 medium containing 100 IU / mL rhIL-2 to make the density 1×10^6 / mL. Add 10 μL of MACS CD3 / CD28 T cell TransAct beads to every 2×10^6 cells. The medium is X-VIVO15 containing 100 IU / mL rhIL-2. Gently mix and culture in a cell incubator. After 24 hours, centrifuge and pour out the supernatant to remove the magnetic beads, resuspend with 1 mL of X-VIVO 15 medium containing 100 IU / mL rhIL-2, add the target lentivirus according to the total number of cells and the measured virus titer (infection at MOI = 10). At the same time, set aside some cells without adding lentivirus as the control group without transduced TCR. Each group was supplemented with culture medium to a final volume of 2 mL of X-VIVO 15 containing 100 IU / mL IL-2, add polybrene at a final concentration of 10 μg / mL, and centrifuge in a plate basket at 37°C and 2000g for 60 minutes. Put the infected cells into a carbon dioxide incubator and culture for 24h. Regularly change the fresh medium and adjust the cell density, and culture until the 14th day.

[0130] The expression rate of TCR-T was detected using GFP. The results showed (see Figure 2 ) that each TCR could be stably expressed in the cell membrane.

[0131] Example 6: Specific IFN-γ and IL-2 secretion of overexpressed TCR-T cells against antigen-positive target cells

[0132] 1. Use TCR-expressing T cells (the same as in Example 5) as effector cells, and use PBMCs not transduced with TCR amplified and cultured in parallel as a control for effector cells.

[0133] 2. Use K562-CD80-HLA-A*02:01 loaded with 10^-7M AFP (FMNKFIYEI) short peptide or an irrelevant peptide as positive target cells (the same as in Example 4); the E:T effector-to-target ratio (effector cell:target cell ratio) is 1:1, and the expression of cell IL-2 or / and IFN-γ is detected after incubation for 24 h.

[0134] 3. Use K562-CD80-HLA-A*02:01 loaded with different concentrations of AFP (FMNKFIYEI) short peptide from 10^-10M to 10^-5M as positive target cells (the same as in Example 4); the E:T effector-to-target ratio (effector cell:target cell ratio) is 1:1, and the expression of cell CD137 is detected after incubation for 24 h.

[0135] The results showed that in the presence of positive target cells, the overexpressed TCR-T group produced IL-2 and IFN-γ and the expression of CD137 was upregulated, and the overexpressed TCR-T group did not produce IFN-γ or IL-2 against negative target cells. Some of the results of IL-2 and INF-γ expression and secretion are shown respectively in Figure 3 and Figure 4 respectively. Some of the results of CD137 upregulation are shown in Figure 5 respectively. The above data indicate that: the TCR-expressing T cells related have specific activation efficacy against antigen-positive target cells, and the TCR protein obtained in the present invention is suitable for use for diagnostic and therapeutic purposes.

[0136] Example 7: Specific killing activity of overexpressed TCR-T cells against antigen-positive tumor cells

[0137] 1. Use TCR-expressing T cells (the same as in Example 5) as effector cells, and use PBMCs not transduced with TCR amplified and cultured in parallel as a control (blank) for effector cells.

[0138] 2. Use 293T-CD80-HLA-A*02:01 (same as Example 5) with AFP (FMNKFIYEI) short peptide at a load of 10^-8M to 10^-6M as the positive target cells (+); the E:T (effector cell:target cell ratio) is 10:1. The adhesion ability of the target cells is detected in real time by an RTCA (Real-Time Cell Analyzer, which integrates a microelectronic cell sensor chip at the bottom of the cell detection plate and can obtain biological information related to cell physiological functions, including cell growth, spreading, morphological changes, death, and adhesion, etc.) instrument. Specifically: every 15 minutes, the instrument collects the cell adhesion ability value (Cell Index) for each well. When processing the subsequent data, the data at the last time point before adding T cells is used as the normalized value, and the normalized cell adhesion ability value (Normalized Cell Index) at each time point for each group is calculated; the results show (see Figure 6 ): The TCR-T overexpression group only has significant killing activity against 293T-CD80-HLA-A*02:01 loaded with tumor-associated antigen polypeptides and has no killing effect on 293T-CD80-HLA-A*02:01 not loaded with polypeptides, among which the killing effect of Y23191 is better.

Claims

1. A T cell receptor targeting an AFP peptide, the sequence of the AFP peptide being FMNKFIYEI, and the T cell receptor comprising a variable domain of the TCRα chain and a variable domain of the TCRβ chain, Among them, The variable domain of the TCRα chain comprises: CDR1α with the sequence DSASNY; CDR2α with the sequence IRSNVGE; and CDR3α with the sequence AAPFGGEAGTALI, wherein, the variable domain of the TCRβ chain comprises: CDR1β with the sequence DFQATT; CDR2β with the sequence SNEGSKA; and CDR3β with the sequence SARGPLIGPTDTQY.

2. The T cell receptor according to claim 1, wherein, The T cell receptor is soluble.

3. The T cell receptor according to claim 1, wherein, There is an artificial disulfide bond between the constant region of the α chain and the constant region of the β chain of the T cell receptor.

4. The T cell receptor according to any one of claims 1 to 3, Among them, The sequence of the variable domain of the TCRα chain is: MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAAPFGGEAGTALIFGKGTTLSVSSN, wherein, the sequence of the variable domain of the TCRβ chain is: MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARGPLIGPTDTQYFGPGTRLTVLE.

5. A synthetic nucleic acid molecule encoding the T cell receptor according to claim 1.

6. A vector containing the synthetic nucleic acid molecule according to claim 5.

7. A host cell containing the vector according to claim 6.

8. A host cell in which the synthetic nucleic acid molecule according to claim 5 is integrated into the chromosome.

9. A method for preparing a T cell receptor, comprising: (i) culturing the host cell according to claim 7 or 8 to express the T cell receptor according to claim 1; and (ii) isolating the T cell receptor.

10. A companion diagnostic kit for tumor immunotherapy, comprising: A T cell receptor protein, the T cell receptor protein comprising a variable domain of the TCRα chain and a variable domain of the TCRβ chain, wherein, the variable domain of the TCRα chain comprises: CDR1α with the sequence DSASNY; CDR2α with the sequence IRSNVGE; and CDR3α with the sequence AAPFGGEAGTALI, wherein, the variable domain of the TCRβ chain comprises: CDR1β with the sequence DFQATT; CDR2β with the sequence SNEGSKA; and CDR3β with the sequence SARGPLIGPTDTQY, The T cell immune receptor targets a tumor antigen peptide derived from the AFP gene, and the sequence of the tumor antigen peptide is: FMNKFIYEI.

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